EP1718973B1 - Method for the rapid diagnosis of targets in human body fluids - Google Patents

Method for the rapid diagnosis of targets in human body fluids Download PDF

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Publication number
EP1718973B1
EP1718973B1 EP05707291A EP05707291A EP1718973B1 EP 1718973 B1 EP1718973 B1 EP 1718973B1 EP 05707291 A EP05707291 A EP 05707291A EP 05707291 A EP05707291 A EP 05707291A EP 1718973 B1 EP1718973 B1 EP 1718973B1
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EP
European Patent Office
Prior art keywords
sample
analysis device
swab member
pathogen
allergy
Prior art date
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Active
Application number
EP05707291A
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German (de)
English (en)
French (fr)
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EP1718973A2 (en
Inventor
Franz Aberl
Marcus Scheibenzuber
Robert P. Sambursky
Robert W. Vandine
Jose S. Sambursky
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Rapid Pathogen Screening Inc
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Rapid Pathogen Screening Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/065—Poxviridae, e.g. avipoxvirus
    • G01N2333/07—Vaccinia virus; Variola virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/811—Test for named disease, body condition or organ function
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
    • Y10T436/255—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.] including use of a solid sorbent, semipermeable membrane, or liquid extraction

Definitions

  • the present invention relates to a method for the detection of targets, e.g. pathogens and/or allergy-associated components in a human eye fluid wherein an eye fluid sample is collected with a swab member.
  • targets e.g. pathogens and/or allergy-associated components in a human eye fluid
  • the samples are directly transferred from the swab member to a sample analysis device, on which an analysis of the targets, e.g. by immunochemical or enzymatic means can take place.
  • the test result may be displayed within a short period of time and can be directly read out by the user.
  • a test kit for carrying out the method of the invention is provided.
  • Rapid, point-of-care analysis is becoming increasingly important in the diagnosis and treatment of various viral and other pathogenic microbiological agents (bacteria, others). Especially in the acute status of a infectious disease medical doctors have a need for immediate detection of the causal agent for the symptoms observed.
  • Prior art discloses a rapid assay for HIV specific antibodies in saliva samples.
  • a saliva sample is gained by means of a sampling stick.
  • the saliva sample is diluted in a sample buffer and a lateral flow immunoassay is dipped into the diluted saliva sample [United States Patent 5,714,341 ].
  • German Patent Nr. DE19622503 suggests to apply lateral flow immunoassays for the detection of illegal narcotics in saliva or sweat.
  • Conjunctivitis commonly known as red eye or pink eye, may be caused by several different agents including viruses, bacteria and allergens. Different etiologies require different treatments. Infectious conjunctivitis is typically contagious. Conjunctivitis is generally diagnosed clinically, by gross examination, and (during a routine eye exam) slit lamp biomicroscopy. This method does not provide information on the specific infectious agent. If specific (pathogen typing) diagnosis is necessary, swabs of the inferior fornix are sent for laboratory analysis to determine the type of pathogen. The preferred methods for laboratory analysis are cell culture with confirmatory direct immunofluorescence, ELISA or PCR.
  • a publication by Uchio et al. discloses a method for the detection of adenovirus in eye fluid specimens.
  • the method comprises collecting a sample of eye fluid and detecting the analyte on a paper disc by enzyme immunoadsorption.
  • the detection however, lacks specificity and sensitivity.
  • the present invention relates to a method for the detection of a target which is selected from pathogens and/or allergy-associated components in an eye fluid comprising the steps:
  • the invention relates to a method for diagnosing conjunctivitis comprising the steps:
  • test kit in the inventive method, wherein the test kit comprises
  • the invention provides a sensitive and rapid method for the detection of targets, e.g. pathogens and/or allergy-associated components in samples collected by non-invasive means from an eye fluid.
  • targets e.g. pathogens and/or allergy-associated components in samples collected by non-invasive means from an eye fluid.
  • the pathogens are selected from viruses, microorganisms, e.g. bacteria and parasites, e.g. amoebae or nematodes.
  • the allergy-associated components are selected from allergens and anti-allergic components.
  • the detection comprises the direct detection of the target, e.g. the pathogen and/or the detection of antibodies against the target, e.g. the pathogen which are present in the fluid sample to be tested.
  • the method comprises a parallel determination of a plurality of targets.
  • the body fluid is an eye fluid from a body surface selected from mucose membrane fluids (of ocular cavities) or tears.
  • results are provided within the medical consultation period, e.g. in few minutes. Preferably, the results are provided in a time period up to 20 minutes, more preferably up to 15 minutes. Also, as the test is noninvasive, it poses very little risk to the patient. Thus the best available treatment can be applied on a timely basis for a specific pathogen.
  • a further advantage over prior art methods is that only a few microliters of sample are required to perform an analysis.
  • the sample is about 0.2 ⁇ l to about 20 ⁇ l and more preferably about 0.5 ⁇ l to about 10 ⁇ l.
  • the invention may be performed by means of a simple test kit. Handling of the test kit does not necessitate additional laboratory equipment, further handling of reagents or instrumentation. Another important advantage of the invention described below is that the detection limit is typically 10 to 100 times lower than currently available diagnostic tests because samples do not require dilution before they are transferred to the analysis device. Therefore the disclosed method has proven to be more sensitive and accurate than methods of the prior art.
  • the invention discloses a non-invasive method for the rapid and point-of-care determination of pathogens from eye fluids.
  • the method is suitable for diagnosis in human beings and animals, e.g. pets or livestock animals.
  • a preferred application is the detection of pathogens in human eye fluid.
  • the pathogen to be detected is a causative agent of conjunctivitis or a plurality of such causative agents.
  • the pathogen is selected from the group of adenoviruses, herpesviruses, chlamydiae, cytomegaloviruses and combinations thereof. More preferably, a plurality of pathogens are detected on a single sample analysis device.
  • the sample analysis device may allow a simultaneous detection of a plurality of pathogens, particularly of at least two, of at least three, of at least four or of at least five pathogens selected from the group consisting of adenoviruses, herpesviruses, chlamydiae, cytomegaloviruses, pseudomonas, streptococci, haemophilus, staphylococci, amoebae, particularly Acanthamoeba and nematodes, particularly Onchocera volvulus. More preferably, the method comprises a simultaneous detection of adenoviruses, herpesviruses, chlamydiae, cytomegaloviruses and Acanthamoeba.
  • the invention provides a non-invasive method for the rapid and point-of-care determination of at least one allergy-associated component, particularly an allergen (e.g. pollen, dust, etc.) and/or an antiallergen, particularly a component which is produced in the body in response to an allergenic challenge (e.g. IgE, histamine, etc.), in an eye fluid as described above.
  • an allergen e.g. pollen, dust, etc.
  • an antiallergen particularly a component which is produced in the body in response to an allergenic challenge (e.g. IgE, histamine, etc.)
  • the invention relates to methods and devices for the diagnosis of allergy-associated components in human eye fluid.
  • the determination of at least one allergy-associated component may be combined with the determination of at least one pathogen as described above.
  • an eye fluid sample is non-invasively collected with a collection device or swab member, respectively.
  • the collection step preferably comprises wiping or dabbing the swab member over a surface of the body containing eye fluid to be tested.
  • the swab member is sterile.
  • the swab member may be dry or pretreated with a fluid before the collection step. For example, using a gentle swirling motion, a sterile swab member may be applied to the body surface or mucous membrane of concern and allowed to capture any pathogens and/or allergy-associated components contained in the eye fluid.
  • the swab member may be a part which is separate from the sample analysis device and the sample is transferred by contacting the sample analysis device with the swab member under conditions wherein at least a part of the sample on the swab member is transferred to the sample analysis device.
  • the swab member is preferably contacted with a sample application zone on the analysis device from which the sample is then directly transferred to the detection zone.
  • the contact preferably comprises fixing the swab member in a contact position with the sample analysis device in which the sample collection zone of the swab member is in direct contact with the sample application zone of the analysis device.
  • the swab member and/or the analysis device preferably comprises fixing means for providing a fixed contact between both parts in a predetermined position.
  • the swab member may be an integrated part of the sample analysis device and the transfer comprises passing at least a part of the sample on the swab member to the detection zone on the sample analysis device.
  • the transfer of the sample from the swab member to the detection zone on the sample analysis device is a direct transfer, i.e. the transfer takes place without pretreatment of the sample on the swab member.
  • the transfer comprises an elution of the sample from the swab member with an elution medium, e.g. a buffer or water.
  • the elution medium may be added from an external source or may be provided e.g. as a reservoir within the analysis device.
  • the transfer is preferably a chromatographic and/or capillary transfer of fluid to the detection zone on the sample analysis device.
  • the sample analysis device comprises a chromatographic test strip, e.g. a lateral flow test strip.
  • the sample analysis device may comprise a sample application zone, a detection zone, optionally a waste zone, optionally a carrier backing, optionally a housing and optionally an opening for result read out.
  • the sample analysis in the detection zone may be carried out by standard means, e.g. by an immunological or enzymatic detection method.
  • the detection method comprises the use of test reagents capable of specifically binding the targets, e.g. pathogens to be tested or antibodies or other receptors against these targets, e.g. pathogens and subsequent visualisation of the bound entity, e.g.
  • the swab member is placed on a In an especially preferred embodiment, the swab member is placed on a lateral flow test strip.
  • the test strip consists of one or several capillary active fleeces or membranes.
  • the detection process will be either started directly with sample transfer or may require an elution medium to be applied for sample analysis. Preferably this elution medium is simple tap water.
  • the chosen elution medium moves towards a detection zone and thereby passes the contact site within the collection device.
  • the analyte is diluted by the elution medium and carried with it to the detection zone. In the detection zone the analyte is determined by qualitative and/or quantitative methods, e.g. in an immunological binding reaction.
  • the test strip can be made of one single chromatographic material, or preferably several capillary active materials made of the same or different materials and fixed on a carrier backing. These materials are in close contact with each other so as to form a transport path along which a liquid driven by capillary forces flows from the start zone, passing the contact site of the swab and the detection zone, towards a waste zone at the other end of the strip.
  • this invention is disclosing a device and test kit for the performance of the described method.
  • a chromatography test strip contains:
  • the specific binding partners for the analytes in the conjugate and the detection zone are monoclonal, polyclonal or recombinant antibodies or fragments of antibodies capable of binding to a pathogen.
  • the specific binding partners may also be antigens capable of binding to antibodies against a pathogen or an allergen.
  • Other types of binding partners are bioorganic macromolecules like aptamers or receptors.
  • the conjugate zone may be located before, within or after the sample application zone, seen in the running direction of the eluent liquid.
  • the test line(s) is(are) located after the conjugate/application zone and the control line(s) is (are) located after the test line. Together, the test line(s) and control line(s) comprise the detection zone.
  • binding partners are present in the different zones.
  • a sandwich immunoassay it is preferred to have a labeled, non-immobilized analyte binding partner in the conjugate zone.
  • the binding partner forms a complex with the analyte which is bound to the immobilized binding partner at the test line.
  • the label of the conjugate binding partner is an optically detectable label. Forming a complex at the test line concentrates and immobilizes the label and the test line gets visible for the bare eye, indicating a positive test result.
  • Particularly preferred are direct labels, and more particularly gold labels which can be best recognized by the bare eye.
  • an electronically photometrical read out device can be used to obtain more precise results and a semi-quantification of the analyte.
  • Other labels may be latex, fluorophores or phosphorophores.
  • a sample may be collected with a sample collection device from the patient's eye by a health care professional.
  • the sample collection device should be wiped or dabbed slightly several times between in the inferior fornix of the lower eye lid. If necessary the collection device may be wet with sterile physiological saline to decrease patient's discomfort. This procedure is well known in the ophthalmology practice as it is necessary for collecting specimens for conventional laboratory analysis.
  • the sample collection device comprises a capillary active material suitable for receiving a body fluid sample.
  • the sample collection material is made out of fibers on the basis of cellulose, polyester, rayon or calcium alginate.
  • the sample collection device can also be designed as a microengineered mechanical structure containing microcapillaries and/or microchannels.
  • the collection device is fixed to the plastic housing containing the test strip ( Figure 4 ) and thereby the collection applicator is slightly pressed on the application zone of the strip. The collection device remains in this position.
  • the sample is taken by a standard swab member as currently used in the physician's office or emergency rooms. This swab member is subsequently pressed into the application zone of the chromatographic test strip by means of an additional device similar to the sample collection unit.
  • the sample is taken by a swab member and the sample collection devices is pressed for only a short time into the application zone of the chromatographic test strip.
  • a short period of time preferably means a time up to 20 seconds, particularly between 0.1 and 10 seconds. A transfer of the sample is happening within the contact period.
  • an elution medium is applied by dipping the absorbent pad into the chromatographic liquid.
  • the absorbent pad is made of a particularly well-absorbing material which delivers the liquid for the immunochemical or enzymatic reactions.
  • Preferred elution media are water or buffer solutions that are conventionally used in immunoassays.
  • the elution medium is contained in a reservoir which may be integrated within the analysis device, e.g. as an ampoule or a blister.
  • the reservoir may be opened by fixing the swab member or sample collection device on the detection part of the device or by additional means.
  • the result can be read out in the detection zone.
  • the result is considered positive when at least a partial area of the test line and the control line shows a color change.
  • test strip The structure of a test strip is depicted in Figure 1 .
  • the polyester fleece for the absorbent pad was manufactured by Binzer, Hatzfeld, Federal Republic of Germany.
  • the fleece is a polyester fleece reinforced with 10% curalon.
  • the thickness ranges 1 and 2 mm, the absorbance capacity is 1800 ml/m 2 .
  • the application/conjugate zone consists of 80 parts polyester and 20 parts viscous staple fibers at a thickness of 0.32 mm and an absorbing capacity of 500 ml/m 2 .
  • the fleece is impregnated with the following solutions and then dried: 100 mmol/l HEPES Buffer, pH 7.5, 100 mol/l NaCl, conjugate of gold particles and anti-Hexon antibodies at a concentration that has an optical density of 10 at 520 nm.
  • Hexon is a protein that is common in the capsid of human adenoviruses.
  • the gold sol was manufactured according to standard procedures ( Fres. Nature Vol. 241, p. 20-22, 1973 ). Conjugation with the antibody was carried out according to prior art procedure ( J. Immunol. Meth. Vol. 34, p. 11-31, 1980 ).
  • the sample application takes place in the application/conjugate zone.
  • the detection zone consists out of a nitrocellulose (NC) membrane with a nominal pore size of 8 ⁇ m and a thickness of 100 ⁇ m produced by Schleicher & Schuell, Germany.
  • the test line contains a Hexon specific antibody (not labeled) which is specific for a different epitope than the antibody immobilized on the gold.
  • the control contains the same antibody than the test line and binds any excess of Hexon specific gold. The control line will appear in any case even if Hexon is not present indicating that the test worked correctly.
  • the chromatographic materials are in communication with each other in order to create a fluid pathway.
  • sample collection material may consist of bibulous material such as highly purified cotton fibers which are fixed to the plastic device by ultrasonic welding.
  • Alternative materials may be polyester, rayon, polyamide or other fibrous polymeric materials.
  • a test kit for the detection of Adenovirus antigen (as described in the example above) was used in the Emergency Room of an Ophthalmologic Hospital to diagnose the clinical picture of a "pink" eye. From every patient which has been tested with the test kit a second sample was taken and analysed in the laboratory.
  • the laboratory reference method used in this study was a combination of cell culture and immunfluorescence (IF) detection ( Rodrigues et al., Ophthalmology, 1979 Mar;86(3):452-64 ) which is the current "laboratory gold standard" for determining the presence of adenovirus in human ocular fluid.
  • IF immunfluorescence

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  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
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  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating Or Analyzing Materials By The Use Of Ultrasonic Waves (AREA)
EP05707291A 2004-02-09 2005-02-09 Method for the rapid diagnosis of targets in human body fluids Active EP1718973B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US54230304P 2004-02-09 2004-02-09
PCT/EP2005/001305 WO2005075982A2 (en) 2004-02-09 2005-02-09 Method for the rapid diagnosis of targets in human body fluids

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EP1718973A2 EP1718973A2 (en) 2006-11-08
EP1718973B1 true EP1718973B1 (en) 2009-09-09

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US (4) US7723124B2 (es)
EP (1) EP1718973B1 (es)
JP (2) JP5630936B2 (es)
AT (1) ATE442590T1 (es)
AU (1) AU2005210742B2 (es)
CA (1) CA2554904C (es)
DE (1) DE602005016527D1 (es)
ES (1) ES2332523T3 (es)
WO (1) WO2005075982A2 (es)

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US8647890B2 (en) 2014-02-11
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